Regulation of S-layer synthesis and secretion in Caulobacter crescentus

Caulobacter crescentus is a non-pathogenic, Gram negative bacterium that forms a surface layer (S-layer) that is composed exclusively of the protein RsaA in a crystalline array. RsaA, which constitutes 10-12% of total cellular protein, is secreted by a robust type I protein transport system in C. crescentus. These features have made this bacterium an attractive candidate for biotechnology applications that require the production of proteins of interest at high levels and purity. In a previous study aimed at determining the maximal protein secretion capability of the type I system in C. crescentus, the overexpression of rsaA did not result in the increased production and secretion of RsaA. However, upon further investigation it was determined that the plasmid used to overexpress rsaA included an extended 5' untranslated region (UTR). The results presented herein suggest that this extended 5' UTR caused a decrease in the half-life of rsaA mRNA to ~19 minutes compared to the ~36 minutes half-life of wild type rsaA mRNA which may in turn explain the lack of increased RsaA production. By contrast, production and secretion of RsaA was significantly increased (2.2 ± 0.1 fold) in C. crescentus transformed with a plasmid containing rsaA without an extended 5' UTR when compared to wild type. Deletion of the outer membrane transporters, RsaFa and RsaFb, prevented the secretion of RsaA and resulted in a significant down-regulation of RsaA production. By using quantitative reverse-transcriptase PCR (qRT-PCR) it was determined that the amount of rsaA mRNA in the transporter deletion mutant was similar to wild type (0.9 ± 0.1-fold of wild type). This suggests that the down-regulation of RsaA observed in these mutants occurred at a posttranscriptional level. Previous experiments showed that recombinant forms of RsaA containing an abundance of positively charged amino acids were not detected at the cell surface, indicating a complete inhibition of secretion. Three dimensional models of RsaFa and RsaFb using the Swiss Model program placed twelve negatively charged amino acids near the entrance of the predicted pore structure on the periplasmic side. In order to test the hypothesis that these negatively charged amino acid residues were inhibiting the secretion of recombinant RsaA, site-directed mutagenesis was used to alter them. However, none of the mutants relieved the inhibition of recombinant RsaA secretion. Moreover, three of the mutants, RsaFb-D395A, RsaFb-E185A/D395A, and RsaFb-D395A/E402A, also inhibited the secretion of wild type RsaA. Taken together, these results demonstrate that RsaA expression can be upregulated and that the type 1 secretion system of C. crescentus can facilitate this increase. In addition, regulation of RsaA can occur at a posttranscriptional level when its secretion is blocked, as is the case in the outer membrane transporter deletion mutants. Furthermore, site-directed mutagenesis suggests a role for negatively charged amino acids in the secretion of S-layer protein in RsaFb but not RsaFa.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat